The assembly dynamics of the actin cytoskeleton is crucial for most morphogenetic processes that occur at both cellular and organism levels in eukaryotes. As a consequence, defects in actin dynamics and organization have been implicated in a variety of human diseases. However, our understanding of how actin is regulated in different physiological processes remains limited due to the extraordinary complexity and dynamic nature of the actin cytoskeletal system. The Arp2/3 complex is thought to be a major key element of actin filament nucleation and the formation of branched dendritic networks at the leading edge of motile cells. The role of Arp2/3 complex in these processes can only be understood through studies of the key molecular steps in the Arp2/3-mediated actin nucleation and branch formation. In this Program Project we aim at understanding the detailed structural mechanisms by which the Arp2/3 complex mediates the formation of actin branch junctions and the role of the Arp2/3 complex in vertebrate cell motility. We have assembled a highly synergistic team of Pis with complimentary expertise to achieve these goals through collaborative efforts. This sub-project (Volkmann lab) will contribute the development and application of a diverse set of computational tools for the reconstruction, analysis, and modeling of structural states and distribution patterns. Using these tools we will combine information from various data sources generated by all members of the Program Project to obtain (i) high-resolution models of the intermediates of the branch formation process and of the fully assembled branch itself;(ii) a dynamic, energetically self-consistent, structural model of the transition pathway from the inactive state of the Arp2/3 complex to the fully assembled branch in the dendritic network, and (iii) structural differences between different cell types and between wild-type cells and defective mutants by multi-dimensional and dynamical characterization of Arp2/3-complex and actin-filament distribution patterns in living eukaryotic cells.